Retrovirus production was performed as described previously11 (link). Briefly, transfections of the retroviral constructs together with pHIT60 (gag-pol) and pHIT123 (env) were performed using Lipofectamine 2000 (Invitrogen). 10 mM sodium butyrate was used for induction. The virus supernatant was collected, filtered through a 0.45 μm filter. For lentivirus, PCD/NL-BH (gag-pol) and pMN-VSV-G (env) were used for virus packaging. The lentivirus was concentrated by centricon centrifugal filters from EMD Millipore (Billerica, MA). For transduction, non-tissue culture treated 6-well plates were coated with 50 μg/ml retronectin (Takara, Madison, WI), and virus was loaded by centrifugation (2000 × g, 90 min at 32 °C). Then virus was discarded and 2 × 106 pre-B cells were transduced per well by centrifugation at 600 × g for 30 minutes. Details of retroviral and lentiviral vectors used were provided in Supplementary Table 5.
Centricon centrifugal filter
Manufactured by Merck Group
Sourced in United States
Centricon Centrifugal filter is a laboratory device used for the concentration and purification of macromolecules, such as proteins, DNA, and RNA, by means of centrifugation. It functions by separating the desired molecules from the solution based on their size and molecular weight through a semi-permeable membrane.
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9 protocols using centricon centrifugal filter
1
Retroviral and Lentiviral Transduction Protocol
2
Retroviral and Lentiviral Transduction Protocol
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Retrovirus production was performed as described previously11 (link). Briefly, transfections of the retroviral constructs together with pHIT60 (gag-pol) and pHIT123 (env) were performed using Lipofectamine 2000 (Invitrogen). 10 mM sodium butyrate was used for induction. The virus supernatant was collected, filtered through a 0.45 μm filter. For lentivirus, PCD/NL-BH (gag-pol) and pMN-VSV-G (env) were used for virus packaging. The lentivirus was concentrated by centricon centrifugal filters from EMD Millipore (Billerica, MA). For transduction, non-tissue culture treated 6-well plates were coated with 50 μg/ml retronectin (Takara, Madison, WI), and virus was loaded by centrifugation (2000 × g, 90 min at 32 °C). Then virus was discarded and 2 × 106 pre-B cells were transduced per well by centrifugation at 600 × g for 30 minutes. Details of retroviral and lentiviral vectors used were provided in Supplementary Table 5.
3
Refolding and Purification of Denatured MICA
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Denatured MICA fractions were pooled and refolded by rapid dilution in 100 mL of renaturing buffer (50 mM Tris, 500 mM NaCl, 3 mM GSH, 0.3 mM GSSG, pH 7.4) in order to achieve a protein concentration <10 µg/mL. Diluted protein was mixed overnight at room temperature. Next, the mix was filtrated through a 0.22 µm syringe filter unit and concentrated using 10 kDa Centricon centrifugal filters (Merck Milipore, Germany) to a final volume <1 mL. Renaturation was verified by size-exclusion chromatography in an Äkta Purifier FPLC with a Superdex 5/150 GL column (GE Healthcare, USA).
4
Gelatin Zymography for Protease Activity
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Confluent PC3 cell cultures were treated with TRI-BE (20% ethanol was used as the vehicle) and only FBS for the positive control. Conditioned (treated) media were collected and concentrated using Centricon centrifugal filters (Millipore) at 2500 rpm for 2 h. Equal volumes of concentrated conditioned medium were mixed with sample buffer (2.5% SDS, 1% sucrose, and 4 µg/mL phenol red) without reducing agents and loaded onto 8% acrylamide gels (30%, 29:1 acrylamide/Bis solution, BIO-RAD) copolymerized with gelatin at 1 mg/mL. The gels were washed twice in 2.5% Triton X-100 and incubated in activation buffer (50 mM Tris-HCl, 5 mM CaCl2, pH 7.4) at 37 °C for 24 h. The gels were fixed and stained with Coomassie Brilliant Blue G-250. Proteolytic activity was detected with the presence of clear bands against the stained background of an undigested substrate. Gels were scanned, and bands were quantified using ImageJ 1.52a.
5
Senescent ESCC cell-derived factors
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ESCC cells were seeded in 6-well plates at a density of 1 × 105 (link) cells per well, cultured overnight, and treated with or without CDDP at the indicated concentrations for 96 h. Then, cells were washed with PBS and cultured in serum-free 1640-RPMI medium. After 48 h, the CM was collected, centrifuged, and filtered to remove cell debris. The CM was concentrated 5-fold with a Centricon Centrifugal filter (3 kD, Millipore, Temecula, CA, USA). The concentration of the CM was normalized to the number of cells collected. The CM from CDDP-treated ESCC cells was defined as Sen CM, and the CM from untreated cells was defined as n-Sen CM. For in vitro co-culture experiments, ESCC cells treated with or without CDDP for 96 h were washed with PBS and co-cultured with or without F. nucleatum (MOI = 100) in antibiotic-free RPMI-1640 medium supplemented with 10% FBS at 37°C under 5% CO2 for 24 h. Then, cells were washed with PBS and cultured in serum-free 1640-RPMI medium for 48 h. The CM from F. nucleatum-infected or uninfected senescent ESCC cells (defined as Sen+F. nucleatum CM or Sen CM) and the CM from F. nucleatum-infected or uninfected non-senescent cells (as n-Sen+F. nucleatum CM or n-Sen CM) were harvested and then used for the following experiments.
6
Conditioned Medium Protein Analysis
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After the cancer cells or single-cell suspensions of lungs had been cultured in serum-free medium for 24 h, the conditioned medium was collected and centrifuged at 1,000 g for 5 min. For western blot analysis, conditioned medium was concentrated about 40-fold using Centricon Centrifugal filter (Millipore, Billerica, MA, USA).
7
Conditioning Media Collection and Preparation
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As previously described, CM was collected after cells had been cultured in serum‐free medium for 24 h and was centrifuged at 1000 g for 5 min [33 (link)]. We collected CM of stable knockdown and overexpression of LAMC1 KYSE30 and KYSE450 cells with or without different treatments [PBS, tumor necrosis factor α (TNF α), MK‐2206, JSH], the CM of CAF cocultured with shLAMC1 and sh‐vector ESCC cells with or without SB225002, and CAF with different intervention conditions (PBS, CM of shLAMC1, recombinant CXCL1, SB225002). The CM was concentrated only 40‐fold for western blotting via a Centricon Centrifugal filter (3 kd, Millipore, Temecula, CA, USA). The CM used to stimulate cells was sterile filtered and diluted once with medium.
8
Conditioned Medium Collection and Preparation
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In brief, the CM was collected after shPLAU-1, shPLAU-2, -vec KYSE-30, and KYSE-450 cells and PLAU, vector KYSE-180 and KYSE-450 cells were cultured in serum-free medium for 24 h. The CM of CAFs was collected as follows. After exposure to various treatments (stimulated by drugs or CM of tumor cells, or cocultured with tumor cells), the medium was replaced with the serum-free medium, and cells were incubated for 24 h. CM was centrifuged at 1000×g for 5 min for further experiments. For western blot, CM was concentrated 40-fold using a Centricon Centrifugal filter (Millipore, USA). For cell stimulation, CM was sterile filtered and diluted once with the medium.
9
Purification of Human Wild-type αSyn Protein
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The construct encoding the full-length human wild-type αSyn inserted in the pET21d plasmid was a kind gift from Brett Lauring (Columbia University, New York, NY). Purification was performed as described previously (Martinez et al., 2003 (link)). Bacteria were induced during the exponential phase with 1 mM isopropyl β-d -1-thiogalactopyranoside for 2 h and harvested by centrifugation. The pellet was solubilized in 20 mM HEPES/KOH, pH 7.2, and 100 mM KCl (buffer A) and heated for 5 min at 90 °C. The cell lysate was centrifuged at 72,000 × g for 30 min, and the supernatant was loaded on a HiTrap monoQ column (GE Healthcare). αSyn was eluted with a liner gradient of KCl from 100 to 500 mM, and the fractions of interest were concentrated using a Centricon centrifugal filter (Millipore) before loading on a Superose 12 column in buffer A (GE Healthcare). The fractions containing αSyn were pooled, concentrated, and stored at − 80 °C. To control αSyn purity, 1 mg of purified Syn was loaded on top of a Superdex 75 10/300 column (GE Healthcare) and eluted at 0.5 ml/min on an AKTA Purifier apparatus. Optical density (OD) was continuously measured at 280 nm. Control samples were incubated with the elution buffer used for αSyn purification.
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